Method for using probe based PCR detection to measure the levels of circulating demethylated beta cell derived DNA as a measure of beta cell loss in diabetes

ABSTRACT

A method for measuring blood levels of β cell DNA that is released upon β cell death by using a quantitative probe technology to detect amplified methylated and demethylated forms of the insulin gene DNA, representing normal tissue and β cell specific origin, respectively. Using probes permits the sensitive and specific identification of demethylated insulin DNA patterns that are present only in β cells. The method offers a bioassay for detecting β cell loss in diabetes, useful for screening of prediabetes, monitoring of disease progression, and selection and monitoring of therapies. The technique finds potential use in both Type I and Type II diabetes, as well as gestational diabetes.

CROSS-REFERENCE TO RELATED APPLICATION

The present application is a Continuation of U.S. patent applicationSer. No. 13/784,017, filed Mar. 4, 2013, now U.S. Pat. No. 9,127,317,issued Sep. 8, 2015, which claims priority to U.S. Provisional patentApplication No. 61/606,140, filed Mar. 2, 2012, and claims benefit ofpriority under 35 U.S.C. 371 from PCT/US13/288862 filed Mar. 4, 2013 (WO2013/131083, published Sep. 6, 2013), entitled METHOD FOR USING PROBEBASED PCR DETECTION TO MEASURE THE LEVELS OF CIRCULATING DEMETHYLATED βCELL DERIVED DNA AS A MEASURE OF β CELL LOSS IN DIABETES, the entirecontent of which are incorporated by reference herein.

BACKGROUND OF THE INVENTION Field

The present application relates to compositions and methods forassessing β cell loss by quantitating extrapancreatic demethylated βcell derived DNA with methylation status-specific oligonucleotide probesthat target Polymerase Chain Reaction (PCR)-amplified DNA sequences ofgenes that are uniquely expressed by β cells.

The loss of insulin producing β cells results in glucose intolerance andthe development of Type 1 (T1D) and Type 2 (T2D) diabetes. Eitan Akirav,Jake A. Kushner and Kevan C. Herold, “β-Cell Mass and Type 1 DiabetesGoing, Going, Gone?”, doi: 10.2337/db07-1817 Diabetes November 2008 vol.57 no. 11 2883-2888. Currently, evaluation of β cell mass is carried outby measuring β cell products such as c-peptide. While useful, thesemeasures do not provide real time information about active β cell loss.

There has thus been a long-felt need for a method capable of accuratelyevaluating β cell death so as to improve disease diagnosis, allow fordisease staging, and provide a better evaluation of clinical treatmentefficacy. There is further a great need in the art for compositions andmethods for non-invasively monitoring β cell destruction in individualshaving, or at risk of developing, diabetes mellitus (“diabetes”),including Type-1 and Type-2 Diabetes (T1D and T2D, respectively), aswell as gestational diabetes.

Epigenetic modifications of DNA control cell-type specific geneexpression. DNA methylation is one example of an epigenetic modificationthat affects gene. Methylation of DNA occurs at CpG dinucleotide sites,and this modification maintains a transcriptionally repressive chromatinconfiguration (Miranda et al., 2007, J. Cell Physiol. 213:384-390).Conversely, demethylation of CpG dinucleotide sites allows atranscriptionally permissive configuration (Id). Beta cells expressinsulin, and thus, maintain a transcriptionally-permissivehypomethylated regulatory region for the insulin gene (INS). Indeed,Genomic DNA sequences near the insulin gene are methylated in non-β cellcell types. Ley, Timothy J., et al. “DNA methylation and regulation ofthe human beta-globin-like genes in mouse erythroleukemia cellscontaining human chromosome 11.” Proceedings of the National Academy ofSciences 81.21 (1984): 6618-6622.) Therefore, the presence ofhypomethylated insulin gene DNA outside of the pancreas of a subjectcorrelated with the release of hypomethylated insulin gene DNA from deadand dying (e.g., apoptotic) β cells. Id. and Kuroda A, Rauch T A,Todorov I, Ku H T, Al-Abdullah I H, et al. (2009) Insulin GeneExpression Is Regulated by DNA Methylation. PLoS ONE 4(9): e6953.doi:10.1371/journal.pone.0006953.

SUMMARY OF THE INVENTION

A method is provided for the detection of extrapancreatic circulating βcell-derived DNA that is indicative of acute and chronic β celldestruction, and thus provides an early biomarker for β cell death inhuman tissues, serum and other bodily fluids, such as saliva. The methodcan identify β cell death before the onset of hyperglycemia anddiabetes. This strategy may prove useful for monitoring β celldestruction in individuals at risk for the development of diabetes,monitoring the progression of β cell destruction in individuals withdiabetes, and use as a marker to guide therapy in patients with diabeteswith possible ongoing β cell destruction.

In various embodiments, methods of the invention assesses the presenceof β cell-derived DNA that is released upon β cell death by using aquantitative probe technology in a traditional PCR assay. By usingprobes, the method permits one to identify demethylated insulin DNApatterns that are present only in β cells. Therefore, the methodprovides a bioassay for detecting β cell loss in diabetes to provide amethod capable of improving disease diagnosis, allowing for diseasestaging, and providing a better evaluation of clinical treatmentefficacy. In various embodiments of the invention detects β cell lossassociated with T1D, T2D, or gestational diabetes, or any combinationthereof.

The method as disclosed herein uses a stepwise detection and analysis ofβ cell and non-β cell derived insulin DNA. The key principle behind themethod is the existence of unique DNA methylation patterns in the βcells that are absent from other cells in the body. That is, the islet βcell DNA methylation pattern associated with the insulin gene isreasonably unique, and the level of islet β cell-origin insulin gene DNAin the serum and other body fluids is altered by islet β cell death orpathology.

By first conducting a bisulfate conversion of DNA extracted from abodily fluid of an individual, it becomes possible to quantify therelative abundance of β cell insulin DNA in the circulation, and hencewhether that individual is experiencing β cell loss.

A method is developed for detecting β cell death in vivo by amplifyingregions of genes that: i) are expressed in β cells(e.g., INS); and ii)contain CpG methylation sites, and then measuring the proportion of βcell-derived DNA in the serum or other body fluids. Generally, by usingprobes that are specific for DNA methylation patterns in β cells,circulating copies of β cell-derived demethylated DNA are detected afterbisulfite treatment and PCR amplification. See, Darst RP, Pardo CE, AiL, Brown KD, Kladde MP; “Bisulfite sequencing of DNA”, Curr Protoc MolBiol. 2010 July; Chapter 7: Unit 7.9.1-17. doi:10.1002/0471142727.mb0709s91; “Methylation Analysis by BisulfiteSequencing: Chemistry, Products and Protocols from Applied Biosystems”,tools•thermofisher•com/content/sfs/manuals/cms 039258/•pdf,www•methods•info/Methods/DNA methylation/Bisulphite sequencing•html eachof which is expressly incorporated herein by reference. The methodprovides a noninvasive approach for detecting β cell death in vivo thatmay be used to track the progression of diabetes and guide itstreatment.

It is likewise understood that specific other tissues and cell types mayhave distinct methylation patterns from other tissues, and thereforethat a corresponding technique, using appropriate PCR primers anddetection probes, may be used to detect apoptosis or other DNA releasefrom these specific tissues or cell types into body fluids.

As an alternate to serum, saliva may also contain sufficient DNAcontaining epigenetic DNA modifications to provide a basis fordiagnosis. During cell death most of the nuclear DNA is converted intonucleosomes and oligomers (Umansky, S. R., et al. [1982], “In vivo DNAdegradation of thymocytes of gamma-irradiated or hydrocortisone-treatedrats”; Biochim. Biophys. Acta 655:9-17), which are finally digested bymacrophages or neighboring cells. However, a portion of this degradedDNA escapes phagocytic metabolism, and can be found in the bloodstream(Lichtenstein, A. V., et al. [2001], “Circulating nucleic acids andapoptosis”; Ann NY Acad Sci, 945:239-249), and also in bodily fluids.The present invention addresses the detection of beta cell-specificepigenetic modifications that are detectable in bodily fluids such asplasma and saliva following the destruction of beta cells.

A method is provided for the sensitive and specific detection of β celldeath in vivo in models of autoimmune and chemically induced diabetes inmice, in human tissues, and in serum from patients with T1D and T2D.This assay identifies a specific methylation pattern in the β cellinsulin DNA. This method provides a biomarker for detecting β cell lossin prediabetic mammals during progression of diabetes.

A preferred method thus comprises the following steps:

1) Serum/plasma, or other body fluid is collected and DNA is extractedand substantially purified. Serum is reasonably available and usable,but collection of saliva may be deemed less invasive.

2) Purified DNA is treated with bisulfite, whereupon the bisulfiteconverts demethylated cytosines to uracil while sparing the methylatedcytosines (see en•wikipedia•org/wiki/Bisulfite sequencing and“Methylation Analysis by Bisulfite Sequencing: Chemistry, Products andProtocols from Applied Biosystems”, Invitrogen Corp. (2007)tools•thermofisher•com/content/sfs/manuals/cms 039258•pdf, expresslyincorporated herein by reference in their entirety; see alsoen•Wikipedia•org/wiki/DNA methylation expressly incorporated herein byreference)(other methylation-sensitive distinctions may be exploited todistinguished between methylated and demethylated DNA, as known in theart).

3) Circulating DNA exists in relatively low abundance. Therefore,bisulfite treated DNA is subject to a 1^(st) step PCR. This reaction ismethylation insensitive and is designed to increase the availability ofDNA template. PCR products are run on a standard gel electrophoresis andpurified. Since the DNA is previously bisulfate treated, there will bedistinct DNA subpopulations corresponding to methylated and demethylatedinsulin gene DNA.

4) Purified DNA is used for a methylation sensitive reaction, that is,the reaction distinguishes between amplified DNA corresponding tomethylated insulin gene DNA and demethylated insulin gene DNA (i.e.,from β cells). The reaction uses methylation sensitive probes to detectand differentiate demethylated insulin DNA from β cell origin frommethylated insulin DNA of non-β cell origin.

Optionally, relative numbers of β cell derived DNA are presented as“methylation index” or 2^((methylated DNA-demethylated DNA)) or thedifference between methylated DNA and demethylated DNA. Otherquantitative analysis of the results, as well as historical trendanalysis is possible. Further, the amount of β cell derived DNA may benormalized on a different basis than non-β cell derived DNA representingthe insulin gene. For example, a tracer similar in characteristics tothe β cell derived DNA (but unique with respect to endogenous DNA) maybe quantitatively injected into a patient.

5) Provide a quantitative reference for the amount of β cell derived DNAnormalized for dilution, degradation, secretion/excretion factors, etc.

It is therefore an object to provide a method for monitoring beta celldeath, comprising: extracting and purifying DNA from a body fluid of ananimal; treating the extracted purified DNA with bisulfite to convertdemethylated cytosine to uracil while sparing the methylated cytosines;amplifying the bisulfite-treated DNA using polymerase chain reaction;purifying the amplified bisulfite-treated DNA; performing a methylationsensitive reaction on the purified bisulfite-treated DNA using at leasttwo different methylation specific probes which quantitativelydistinguish between demethylated insulin DNA of β cell origin andmethylated insulin DNA of non-β cell origin; and computing aquantitative relationship between methylated insulin DNA anddemethylated insulin DNA.

It is a further object to provide a method for monitoring cell death ofa cell type having at least one DNA portion that has a unique DNA CpGmethylation pattern as compared to other cells, which is released intobody fluids upon cell death of cells of the cell type, comprising:extracting and purifying DNA that comprises the DNA portion; treatingthe extracted purified DNA with bisulfite to convert cytosine to uracilwhile sparing the CpG methylated cytosines; amplifying a region of thebisulfite-treated DNA that comprises the DNA portion by polymerase chainreaction using DNA CpG methylation pattern independent primers;determining a quantitative relationship between the DNA portion havingthe unique DNA CpG methylation pattern to the DNA portion lacking theunique DNA CpG methylation pattern, by employing the DNA CpG methylationpattern-specific probes; computing a difference between the DNA portionhaving the unique DNA CpG methylation pattern and the DNA portionlacking the unique DNA CpG methylation pattern.

Another object provides a method for monitoring beta cell death,comprising: extracting and purifying genomic DNA from a body fluid of ananimal, wherein the genomic DNA comprises at least a portion of a genethat is predominantly expressed by β cells and that contains a CpGmethylation site; treating the genomic DNA with bisulfite; performing apolymerase chain reaction (PCR) with primers that flank a region of thegenomic DNA that comprises the CpG methylation site; purifying the PCRproducts; melting the PCR products into single strands; hybridizing thesingle-stranded PCR products with a first oligonucleotide probe capableof hybridizing with a target sequence that comprises a sitecorresponding to a bisulfite-converted CpG site and a secondoligonucleotide probe capable of hybridizing with a target sequence thatcomprises a site corresponding to a bisulfite-nonconverted CpG site, andwherein the probes each comprise a non-FRET label pair consisting of afluorophore and a quencher, and wherein interaction of the firstoligopeptide probe or second oligopeptide probe with a respective targetcauses the first oligopeptide probe or second oligopeptide probe tochange from a first conformation to a second conformation, therebychanging the distance between the fluorophore and quencher of said labelpair, and wherein in only one conformation do the fluorophore andquencher interact sufficiently to quench the fluorescence of thefluorophore by a predetermined amount; quantitatively measuringfluorescent signals emitted by the first oligopeptide probe and thesecond oligopeptide probe; and reporting a quantitative relationship ofthe fluorescent signal emitted by the first oligopeptide probe and thesecond oligopeptide probe, indicative of the relative amount of βcell-derived DNA versus non-β cell-derived DNA.

It is also an object to provide a kit for detecting β cell-deriveddemethylated genomic DNA in a biological sample, wherein the kitcomprises: PCR primers that flank a portion of a gene that ispredominantly expressed by β cells and contains a CpG methylation site;a first oligonucleotide probe capable of hybridizing with a first targetsequence on a PCR product made using the PCR primers, wherein the firsttarget sequence corresponds to at least one bisulfite-converted CpG siteof the portion of the gene; and a second oligonucleotide probe capableof hybridizing with a target sequence on a PCR product made using thePCR primers of the kit, wherein the target sequence corresponds to atleast one bisulfite-nonconverted CpG site of the portion of the gene,wherein the first oligopeptide probe and the first oligopeptide probeeach comprise label that allows selective quantitation of the firstoligopeptide probe and the second oligopeptide probe. Each probe maycomprise a label pair consisting of a fluorophore and a quencher, andwherein a binding interaction of the first oligopeptide probe with thefirst target sequence, and the second oligopeptide probe with the secondtarget sequence, causes a change from a first conformation to a secondconformation, thereby changing an interaction between the fluorophoreand quencher of said label pair, and wherein in only one conformation ofthe first and second conformations do the labels interact sufficientlyto quench the fluorescence of the fluorophore by at least 25 percent.

The probes may be conjugated to a fluorophore and/or a quencher. Thefluorophore may be at least one of 6-carboxy fluorescein andtetrachlorofluorescein. The quencher may be tetramethylrhodamine. Theprobe may employ a fluorescent resonant energy transfer (FRET)interaction between the fluorophore and quencher, wherein thefluorophore and quencher are selectively separated in dependence on abinding of the probe to a respective target. The probe may also employ anon-FRET interaction between the fluorophore and quencher, wherein thefluorophore and quencher have an interaction based on a conformation ofthe probe, and in which the conformation is selectively dependent on abinding of the probe to a respective target.

The methylation sensitive reaction may comprises quantitativelydetermining a release of a fluorophore from a probe bound to thepurified bisulfite-treated DNA.

The DNA portion having the unique DNA CpG methylation pattern maycomprise an insulin gene from a pancreatic beta cell. The body fluid maybe, for example, blood, blood plasma, blood serum, saliva, or tears.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1 sets forth the overall procedure for detecting circulating β cellDNA;

FIG. 2A shows the results of testing logarithmic serial dilutions ofsynthetic hypomethylated and hypermethylated DNA;

FIG. 2B shows that Log₁₀ transformation of demethylation indexmeasurements show a non-linear fit;

FIG. 2C shows the increase in specificity and sensitivity of the assayused in the present method;

FIG. 3A demonstrates improved glucose levels in patients withlong-standing Type 2 diabetes;

FIG. 3B shows that the probes according to the present technology reveala significant increase in demethylated β cell DNA in the serum of thepatients with long-standing Type 2 diabetes;

FIG. 3C shows that nested PCR using primers generally according toAkirav (2011) fail to reveal a significant increase in demethylated βcell DNA in the serum of the patients with long-standing Type 2diabetes;

FIG. 4A shows the ability of the assay used in the present method todetect elevated demethylated DNA levels in the ob/ob leptin deficientmouse model of Type 2 diabetes;

FIG. 4B correlates the levels shown in FIG. 4A with elevated bodyweight; and

FIG. 4C correlates the levels shown in FIG. 4A with increased glucoselevels.

DETAILED DESCRIPTION

In various embodiments, the present technology substantially isolatesnucleic acids from a sample of body fluid, for example blood plasma,saliva, spinal fluid, lymph fluid, synovial fluid, or tears, forexample.

Various DNA extraction, isolation and purification technologies can beused, for example as taught in U.S. Pat. Nos. 4,935,342, 5,990,301,6,020,124, 7,241,596, 6,485,903, 6,214,979, Re. 39,920 each of which isexpressly incorporated herein by reference in its entirety.

An anion exchange material may be selected and employed whicheffectively adsorbs the target nucleic acids or protein complexesthereof. For example, commercially available anion exchange materialsmay be employed. Either strong or weak anion exchangers may be employed.A preferred weak exchanger can be one in which primary, secondary, ortertiary amine groups (i.e., protonatable amines) provide the exchangesites. The strong base anion exchanger has quaternary ammonium groups(i.e., not protonatable and always positively charged) as the exchangesites. Both exchangers can be selected in relation to their respectiveabsorption and elution ionic strengths and/or pH for the nucleic acidbeing separated. Purification by anion exchange chromatography isdescribed in U.S. Pat. No. 5,057,426 (see also EP 0 268 946 B1),expressly incorporated by reference herein in its entirety.

The material which is commercially available under the designationQ-Sepharose™ (GE Healthcare) is a particularly suitable. Q-Sepharose™,can be a strong anion exchanger based on a highly cross-linked, beadformed 6% agarose matrix, with a mean particle size of 90 μm. TheQ-Sepharose™ can be stable in all commonly used aqueous buffers with therecommended pH of 2-12 and recommended working flow rate of 300-500cm/h. In other preferred embodiments, the anion-exchange medium can beselected from sepharose-based quaternary ammonium anion exchange mediumsuch as Q-filters or Q-resin.

The chromatographic support material for the anion charge used in theinstant methods can be a modified porous inorganic material. Asinorganic support materials, there may be used materials such as silicagel, diatomaceous earth, glass, aluminum oxides, titanium oxides,zirconium oxides, hydroxyapatite, and as organic support materials, suchas dextran, agarose, acrylic amide, polystyrene resins, or copolymers ofthe monomeric building blocks of the polymers mentioned.

The nucleic acids can also be purified by anion exchange materials basedon polystyrene/DVB, such as Poros 20 for medium pressure chromatography,Poros™ 50 HQ, of the firm of BioPerseptive, Cambridge, U.S.A., or overDEAE Sepharose™ DEAE Sephadex™ of the firm of Pharmacia, Sweden; DEAESpherodex™, DEAE Spherosil™, of the firm of Biosepra, France.

A body fluid sample, such as blood plasma or saliva, containing nucleicacids or their proteinous complexes, is applied to the selected anionexchange material, and the nucleic acids or their complexes becomeadsorbed to the column material.

The contact and subsequent adsorption onto the resin can take place bysimple mixing of the anion exchange media with the body fluid, with theoptional addition of a solvent, buffer or other diluent, in a suitablesample container such as a glass or plastic tube, or vessel commonlyused for handling biological specimens. This simple mixing referred toas batch processing, can be allowed to take place for a period of timesufficiently long enough to allow for binding of the nucleoprotein tothe media, preferably 10 to 40 min. The media/complex can then beseparated from the remainder of the sample/liquid by decanting,centrifugation, filtration or other mechanical means.

The anion exchange material can optionally be washed with an aqueoussolution of a salt at which the nucleic acids remain bound to the anionexchange material, the washing being of sufficient volume and ionicstrength to wash the non-binding or weakly binding components throughthe anion-exchange material. In some embodiments, the resin can bewashed with 2×SSC (300 mM NaCl/30 mM sodium citrate (pH 7.0). Preferredranges of the salt solutions are 300-600 nM NaCl/30 mM sodium citrate(pH 7.0). The resin may alternately be washed with 300-600 mM LiCl/10 mMNaOAc (pH 5.2).

The bound nucleic acids may then be eluted by passing an aqueoussolution through the anion exchange material of increasing ionicstrength to remove in succession proteins that are not bound or areweakly bound to the anion-exchange material and the nucleic acids ofincreasing molecular weight from the column. Both proteins and high andlow molecular weight nucleic acids (as low as 10 base pairs) can beselectively eluted from the resin stepwise with the salt solution ofconcentrations from 300 mM to 2.0 M of NaCl and finally with 2.0 Mguanidine isothiocyanate. LiCl solutions in the concentration range of300 mM to 2.0 M of LiCl may also be used for stepwise elution.

The nucleic acids isolated may be in double-stranded or single-strandedform.

The body fluid can be pre-filtered through a membrane and supplementedwith 10 mM EDTA (pH 8.0) and 10 mM Tris-HCL (pH 8.0) prior to adsorptiononto the anion-exchange medium. Commercial sources for filtrationdevices include Pall-Filtron (Northborough, Mass.), Millipore (Bedford,Mass.), and Amicon (Danvers, Mass.). Filtration devices which may beused are, for example, a flat plate device, spiral wound cartridge,hollow fiber, tubular or single sheet device, open-channel device, etc.

The surface area of the filtration membrane used can depend on theamount of nucleic acid to be purified. The membrane may be of alow-binding material to minimize adsorptive losses and is preferablydurable, cleanable, and chemically compatible with the buffers to beused. A number of suitable membranes are commercially available,including, e.g., cellulose acetate, polysulfone, polyethersulfone, andpolyvinylidene difluoride. Preferably, the membrane material ispolysulfone or polyethersulfone.

The body fluid, for example blood plasma or saliva, can be supplementedwith EDTA and Tris-HCL buffer (pH 8.0) and digested with proteinases,such as for example Proteinase K, prior to adsorption onto the anionexchange medium.

The anion-exchange medium can be immobilized on an individualizedcarrier such as a column, cartridge or portable filtering system whichcan be used for transport or storage of the medium/nucleoprotein boundcomplex. The nucleic acid/anion exchange may be maintained in storagefor up to 3 weeks.

A kit may be provided with a solid carrier capable of adsorbing thenucleic acids containing in a sample of a body fluid, for example bloodplasma or saliva. The kit may also contain other components for example,reagents, in concentrated or final dilution form, chromatographicmaterials for the separation of the nucleic acids, aqueous solutions(buffers, optionally also in concentrated form for final adjusting bythe user) or chromatographic materials for desalting nucleic acids whichhave been eluted with sodium chloride.

The kit may also contain additional materials for purifying nucleicacids, for example, inorganic and/or organic carriers and optionallysolutions, excipients and/or accessories. Such agents are known and arecommercially available. For solid phase nucleic acid isolation methods,many solid supports have been used including membrane filters, magneticbeads, metal oxides, and latex particles. Widely used solid supportsinclude silica-based particles (see, e.g., U.S. Pub. Pat. App.2007/0043216 (Bair Jr., et al.); U.S. Pat. No. 5,234,809 (Boom et al.);WO 95/01359 (Colpan et al.); U.S. Pat. No. 5,405,951 (Woodard); WO95/02049 (Jones); WO 92/07863 (Qiagen GmbH), each of which is expresslyincorporated herein by reference). Inorganic components of carriers maybe, for example, porous or non-porous metal oxides or mixed metaloxides, e.g. aluminum oxide, titanium dioxide, iron oxide or zirconiumdioxide, silica gels, materials based on glass, e.g. modified orunmodified glass particles or ground glass, quartz, zeolite or mixturesof one or more of the above-mentioned substances. On the other hand, thecarrier may also contain organic ingredients which may be selected, forexample, from latex particles optionally modified with functionalgroups, synthetic polymers such as polyethylene, polypropylene,polyvinylidene fluoride, particularly ultra high molecular polyethyleneor HD-polyethylene, or mixtures of one or more of the above-mentionedsubstances.

In addition, the reagent kit may also contain excipients such as, forexample, a protease such as proteinase K, or enzymes and other agentsfor manipulating nucleic acids, e.g. at least one amplification primer,and enzymes suitable for amplifying nucleic acids, e.g. DNase, a nucleicacid polymerase and/or at least one restriction endonuclease.Alternately, a commercial polymerase chain reaction kit may be used toamplify the DNA samples, as discussed below. DNA is subject todegradation by DNases present in bodily fluids, such as saliva. Thus, incertain embodiments, it is advantageous to inhibit DNase activity toprevent or reduce the degradation of DNA so that sufficiently largesequences are available for detection.

After collection, the sample may be treated using one or more methods ofinhibiting DNase activity, such as use of ethylenediaminetetraaceticacid (EDTA), guanidine-HCl, GITC (Guanidine isothiocyanate),N-lauroylsarcosine, Na-dodecylsulphate (SDS), high salt concentrationand heat inactivation of DNase.

After collection, the sample may be treated with an adsorbent that trapsDNA, after which the adsorbent is removed from the sample, rinsed andtreated to release the trapped DNA for detection and analysis. This notonly isolates DNA from the sample, but, some adsorbents, such as Hybond™N membranes (Amersham Pharmacia Biotech Ltd., Piscataway, N.J.) protectthe DNA from degradation by DNase activity.

In some cases, the amount of DNA in a sample is limited. Therefore, forcertain applications, sensitivity of detection may be increased by knownmethods.

Where DNA is present in minute amounts, larger samples can be collectedand thereafter concentrated such as by butanol concentration orconcentration using Sephadex™ G-25 (Pharmacia Biotech, Inc., PiscatawayN.J.).

Once obtained, the bodily fluid derived DNA may be used as an alternateto serum-derived DNA as discussed below. Since the technology isratiometric, it is dependent not on the absolute quantity of DNAavailable, but the proportional relationships of the methylated andunmethylated portions. In general, the disposition of these types in thevarious body fluids is not believed to be highly dependent on the fluidtype, and calibration techniques can be used to account for persistentand predictable differences in the fluid methylated/unmethylated ratios.

Bisulfite Treatment

DNA from serum samples was purified using the Qiagen QIAamp DNA BloodKit following the manufacturer-recommended protocol. Syntheticunmethylated and methylated DNA was purchased from Zymo research. DNAwas then subjected to bisulfite treatment and purified on a DNA bindingcolumn to remove excessive bisulfite reagent using the Zymo EZ DNAMethylation Kit.

First-Step PCR and Gel Extraction.

A methylation-independent reaction was carried out to increase the DNAtemplate for PCR analysis.

For the reaction, bisulfite-treated DNA template was added to Zymo TaqPremix. The amplification proceeded for, e.g., 50 cycles. The PCRproducts were excised from a 3% agarose gel. Negative controls withoutDNA did not yield products in the first-step reaction.

PCR products obtained using methylation-independent primers werepurified using a Qiagen PCR Purification Kit.

Methylation-specific Analysis

Methylation-specific DNA probes are used for the detection of β cellderived DNA. These probes are able to quantitatively and sensitivelydetect circulation demethylated and methylated DNA from a β cell and anon-β cell origin, respectively. The new probes replace the previouslypublished methylation specific primers (see Akirav E M, Lebastchi J,Galvan E M, Henegariu O, Akirav M, Ablamunits V, Lizardi P M, and HeroldK C. Detection of β cell death in diabetes using differentiallymethylated circulating DNA. PNAS, 2011, Proceedings of the NationalAcademy of Sciences, 2011, November: 108(19018-23), expresslyincorporated herein by reference, hereinafter Akirav et al. (2011). Seealso Husseiny M I, Kuroda A, Kaye A N, Nair I, Kandeel F, et al. (2012)Development of a Quantitative Methylation-Specific Polymerase ChainReaction Method for Monitoring Beta Cell Death in Type 1 Diabetes. PLoSONE 7(10): e47942. doi:10.1371/journal.pone.0047942, expresslyincorporated herein by reference), which presented with a relatively lowspecificity (i.e. demethylated primers detected methylated DNA and viceversa). Low specificity negatively impacts assay sensitivity by decreasedetection limits of β cell derived demethylated DNA. Low DNA levels arepresumably present during early β cell loss, such as prediabetes. See,U.S. Pat. No. 6,150,097, expressly incorporated herein by reference.

The overall procedure for the detection of circulating β cell DNA isdepicted in FIG. 1. The steps leading to the use of probes are identicalwith those described in Akirav et al. (2011), which discloses the use ofmethylation-specific primers (and not probes) to detect β cell derivedDNA. The primers were able to detect demethylated and methylated DNAfrom a β cell and a non-β cell origin, respectively. While useful, theseprimers had a relatively low specificity whereby demethylated primersdetected methylated DNA and vice versa. Low specificity reduced assaysensitivity as it impaired the ability to detect very low levels of βcell-derived DNA, such as in the condition of early β cell loss andpre-diabetes.

DNA from serum samples was purified using the Qiagen QIAamp DNA BloodKit following the manufacturer-recommended protocol. Syntheticunmethylated and methylated DNA was purchased from Millipore. PurifiedDNA was quantitated using a NanoDrop 2000 spectrophotometer. DNA wasthen subjected to bisulfite treatment and purified on a DNA bindingcolumn to remove excessive bisulfite reagent using the Zymo EZ DNAMethylation Kit.

The present method, in contrast, uses probe DNA that offers asignificant improvement in sensitivity over the primers used in theprior art discussed above. That is, probe DNA allows for a highlyspecific recognition of two demethylated sites in the insulin gene. Thistends to eliminate false positive readings and thus provides increasedassay specificity and sensitivity. The following is used as probe forthe detection of circulating DNA in the assay according to the presentmethod:

A methylation-independent reaction was carried out to increase the DNAtemplate for PCR analysis. For the reaction, bisulfite-treated DNAtemplate was added to ZymoTaq™ Premix (see,www•zymoresearch•com/protein/enzymes/zymotaq-dna-polymerase, expresslyincorporated herein by reference.) The following PCR primers are used toamplify the human insulin position 2122220-2121985 on chromosome 11,GRCh37.p10, October 2012):

Forward primer: SEQ ID NO: 001 GTGCGGTTTATATTTGGTGGAAGTT Reverse primer:SEQ ID NO: 002 ACAACAATAAACAATTAACTCACCCTACAA

Using the forward and reverse primers, PCR was conducted The PCRproducts were excised from a 3% agarose gel.

The PCR product (or amplicon) is detect by methylation status specificprobes as follows:

a) Probes for the detection of methylated insulin DNA (i.e., DNA notderived from a β cell)(alternates):

SEQ ID NO: 003 ACCTCCCGACGAATCT SEQ ID NO: 004 TACCTCTCGTCGAATCT

b) Probes for the detection of demethylated insulin DNA (i.e., DNAderived from a β cell)(alternates):

SEQ ID NO: 005 ACCTCCCAACAAATCT SEQ ID NO: 006 TACCTCCCATCAAATCT

In various embodiments, the methylation status-specific probes areconjugated with 6-carboxyfluorescein, abbreviated as FAM, thuspermitting quantitative detection. See, en•wikipedia•org/wiki/TaqMan,expressly incorporated herein by reference. Other technologies may beused I conjunction with the present method; see, U.S. Pat. Nos.6,103,476, 8,247,171, 8,211,644, 8,133,984, 8,093,003, 8,071,734,7,972,786, 7,968,289, 7,892,741, 7,847,076, 7,842,811 7,803,528,7,776,529, 7,662,550, 7,632,642, 7,619,059, 7,598,390, 7,422,852,7,413,708, 7,399,591, 7,271,265, 7,241,596, 7,183,052, 7,153,654,7,081,336, 7,070,933, 7,015,317, 7,005,265, 6,811,973, 6,680,377,6,649,349, 6,548,254, 6,485,903, 6,485,901, each of which is expresslyincorporated in its entirety. Probes may be Fluorescent Resonance EnergyTransfer (FRET) or non-FRET type. See, U.S. Pat. No. 6,150,097,expressly incorporated herein by reference. U.S. Pat. No. 6,103,476states: “The use of probes of this invention with interactive labels inassays for the identification of products of nucleic acid amplificationreactions generally eliminates the need for post-amplification analysisto detect desired products and distinguish desired products fromunwanted side reactions or background products. Of course, probesaccording to the invention can be added at the end of a synthesisprocess for end-point detection of products. In assays for monitoringthe progress of an amplification reaction, the probes can be presentduring synthesis. The presence of probes improves the accuracy,precision and dynamic range of the estimates of the target nucleic acidconcentration. Reactions in closed tubes may be monitored without everopening the tubes. Therefore, assays using these probes with interactivelabels can limit the number of false positives, because contaminationcan be limited.”

c) PCR is done with an annealing temperature of 60° C. for 50 cycles andquantified using a Real Time PCR machine. A range of 52-65° C. for thePCR would be acceptable.

d) Values generated by demethylated probes are subtracted from values ofmethylated probes and a delta calculated.

Probe testing of logarithmic serial dilutions of synthetichypomethylated and hypermethylated DNA has shown a linear behavior(R²=0.98) of the delta between hypermethylated DNA and hypomethylatedDNA (delta=hypermethylated DNA-hypomethylated DNA) over a wide range ofDNA dilution (range is 4 log scale) see FIG. 2A. Log₁₀ transformation ofdemethylation index measures show a nonlinear fit (R²=0.9999, DF 2) seeFIG. 2B. FIG. 2C shows the specificity of the assay. The probe detectsdemethylated DNA at ˜180 folds in islet (where β cells reside) comparedwith liver and kidney which do not express insulin. In contrast, primersdetect the demethylated DNA at ˜80 fold. In other words probes usedaccording to an embodiment of the present invention are 2.25 times morespecific than primers the primers used in accordance with Akirav et al.(2011).

The present method extends the use of demethylated β cell derived DNA asa biomarker of Type 2 diabetes. The ability of the present assay todetect β cell loss in Type 2 diabetes is clearly shown by theexperimental results obtained with the use of the present method. FIG.3A shows impaired glucose levels in patients with long-standing Type 2diabetes. FIG. 3B shows the increase in demethylated β cell DNA (i.e.,increase in methylation index) in the serum of these patients, revealedas a significant difference (p=0.0286) from control by the use of thepresent probe technology. Similar results are also observed in animalmodels of Type 2 diabetes. FIG. 3C shows the use of primers from Akiravet al. (2011) to analyze the same sample set, and failed to detect anysignificant difference (p=0.87) in methylation index between control andT2D patients.

For PCR according to Akirav et al., (2011), shown in FIG. 3C wasconducted for 40 cycles, with a melting temperature of 54° C., usingprimers as follows:

Forward primer: SEQ ID NO: 007 TTAGGGGTTTTAAGGTAGGGTATTTGGTReverse primer: SEQ ID NO: 008 ACCAAAAACAACAATAAACAATTAACTCACCCTACAA

The second step real-time methylation-specific nested PCR according toAkirav et al. (2011) was conducted with 50 cycles of amplification, anda melting temperature of 64° C., with the following primers:

Methylated forward primer: SEQ ID NO: 009 GTGGATGCGTTTTTTGTTTTTGTTGGCMethylated reverse primer: SEQ ID NO: 010 CACCCTACAAATCCTCTACCTCCCGDemethylated forward primer: SEQ ID NO. 011TTGTGGATGTGTTTTTTGTTTTTGTTGGT Demethylated reverse primer:SEQ ID NO: 012 CACCCTACAAATCCTCTACCTCCCA

FIG. 4A shows the ability of to detect elevated demethylated DNA levelsin the ob/ob leptin deficient mouse model of type 2 diabetes. Theselevels were correlated with elevated body weight, shown in FIG. 4B, andincreased glucose levels, shown in FIG. 4C.

Although the present invention has been described in relation toparticular embodiments thereof, many other variations and modificationsand other uses will become apparent to those skilled in the art. It ispreferred, therefore, that the present invention be limited not by thespecific disclosure herein, but only by the appended claim.

REFERENCES Each of which is Expressly Incorporated Herein by Reference

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SEQUENCE LISTING APPENDIX <210> 1 <211> 25 <212> DNA <213>Artificial Sequence <220> <223> Forward PCR Primer for insulin gene<400> 1 gtgcggttta tatttggtgg aagtt 25 <210> 2 <211> 30 <212> DNA <213>Artificial Sequence <220> <223> Reverse PCR primer for insulin gene<400> 2 acaacaataa acaattaact caccctacaa 30 <210> 3 <211> 16 <212> DNA<213> Artificial Sequence <220> <223>Probe for the detection of methylated non-beta cell insulin DNA <400> 3acctcccgac gaatct 16 <210> 4 <211> 17 <212> DNA <213>Artificial Sequence <220> <223> Probe for the detection of methylatednon-beta cell insulin DNA <400> 4 tacctctcgt cgaatct 17 <210> 5 <211> 16<212> DNA <213> Artificial Sequence <220> <223>Probe for the detection of demethylated beta cell insulin DNA <400> 5acctcccaac aaatct 16 <210> 6 <211> 17 <212> DNA <213>Artificial Sequence <220> <223> probe for the detection of demethylatedbeta cell insulin dna <400> 6 tacctcccat caaatct 17 <210> 7 <211> 28<212> DNA <213> Artificial Sequence <220> <223>Outer Forward PCR primer for nested PCR according to Akirav etal. (2011) <400> 7 ttaggggttt taaggtaggg tatttggt 28 <210> 8 <211> 37<212> DNA <213> Artificial Sequence <220> <223>Outer Reverse PCR primer for nested PCR according to Akirav etal. (2011) <400> 8 accaaaaaca acaataaaca attaactcac cctacaa 37 <210> 9<211> 27 <212> DNA <213> Artificial Sequence <220> <223>Inner Methylated Forward PCR primer for nested PCR according toAkirav et al. (2011) <400> 9 gtggatgcgt tttttgtttt tgttggc 27 <210> 10<211> 25 <212> DNA <213> Artificial Sequence <220> <223>Inner Methylated Reverse PCR primer for nested PCR according toAkirav et al. (2011) <400> 10 caccctacaa atcctctacc tcccg 25 <210> 11<211> 29 <212> DNA <213> Artificial Sequence <220> <223>Inner Demethylated Forward PCR primer for nested PCR according toAkirav et al. (2011) <400> 11 ttgtggatgt gttttttgtt tttgttggt 29 <210>12 <211> 25 <212> DNA <213> Artificial Sequence <220> <223>Inner Demethylated Reverse PCR primer for nested PCR according toAkirav et al. (2011) <400> 12 caccctacaa atcctctacc tccca 25

What is claimed is:
 1. A method, comprising: receiving from an organisma sample of a circulating, excreted, or secreted body fluid, containingDNA, wherein the sample comprises DNA from apoptosis of pancreatic betacells that give a cell-type-specific demethylation pattern, and DNA fromother corresponding cells not having the cell-type-specificdemethylation pattern; treating the received DNA to distinguish betweenthe DNA having the cell-type-specific demethylation pattern and the DNAnot having the cell-type-specific demethylation pattern, to produce atleast two different DNA products; amplifying the at least two differentDNA products in a quantitative manner by real-time PCR to produce atleast two different amplified DNA products having a corresponding ratioof the DNA from cells having the cell-type-specific demethylationpattern to the other corresponding DNA from cells not having thecell-type-specific demethylation pattern; selectively binding in aquantitative manner, during the real-time PCR, a plurality of differentDNA oligonucleotide probes, each respective probe comprising afluorophore and a quencher, to regions of the at least two differentamplified DNA products which are respectively different dependent on thecell-type-specific demethylation pattern; measuring optical propertiesof the plurality of different DNA oligonucleotide probes during thereal-time PCR, dependent on a change in interaction between thefluorophore and the quencher due to the selectively binding; anddetermining, based on quantitative measurements of the measuredproperties of the plurality of different DNA oligonucleotide probesdependent on the selective binding to regions of the at least twodifferent amplified DNA products, a ratio of the DNA from cells havingan cell-type-specific demethylation pattern to the other correspondingDNA from cells not having the cell-type-specific demethylation pattern.2. The method according to claim 1, wherein the sample is derived fromat least one of blood plasma, saliva, spinal fluid, lymph fluid,synovial fluid, and tears.
 3. The method according to claim 1, whereinthe sample is a human sample derived from at least one of blood plasma,saliva, spinal fluid, lymph fluid, synovial fluid, and tears.
 4. Themethod according to claim 1, wherein said treating the received DNAcomprises purifying DNA contained in the sample and treating thepurified DNA with bisulfite to convert demethylated cytosine to uracilwhile sparing the methylated cytosines.
 5. The method according to claim1, wherein the polymerase chain reaction is conducted using:a forward primer: SEQ ID NO: 001 GTGCGGTTTATATTTGGTGGAAGTT; anda reverse primer: SEQ ID NO: 002 ACAACAATAAACAATTAACTCACCCTACAA.


6. The method according to claim 1, wherein the plurality of differentDNA oligonucleotide probes is selected from the group consisting of:SEQ ID NO: 003 ACCTCCCGACGAATCT; SEQ ID NO: 004 TACCTCTCGTCGAATCT;SEQ ID NO: 005 ACCTCCCAACAAATCT; and SEQ ID NO: 006 TACCTCCCATCAAATCT.


7. The method according to claim 1, wherein the plurality of differentDNA oligonucleotide probes comprises: a) at least one probe fordetection of methylated insulin DNA of non-pancreatic beta cell originselected from the group consisting of: SEQ ID NO: 3 ACCTCCCGACGAATCT;and SEQ ID NO: 4 TACCTCTCGTCGAATCT; and

b) at least one probe for detection of demethylated insulin DNA ofpancreatic beta cell origin selected from the group consisting of:SEQ ID NO: 005 ACCTCCCAACAAATCT; and SEQ ID NO: 006 TACCTCCCATCAAATCT.


8. A method, comprising: receiving from a human a sample of acirculating, excreted, or secreted body fluid containing DNA, at least aportion of the DNA having a beta cell-specific demethylation pattern ofan insulin gene, wherein the sample comprises DNA from the apoptosis ofbeta cells, and DNA from other cells; extracting DNA from the sample;treating the extracted DNA with bisulfite to distinguish between DNAhaving the beta cell specific demethylation pattern and DNA not havingthe beta cell specific demethylation pattern by converting demethylatedcytosine of beta cells having the beta cell specific demethylationpattern to uracil while sparing methylated cytosines of non-beta cellsnot having the beta cell specific demethylation pattern; amplifying thetreated DNA products corresponding to the insulin gene, in a real-timepolymerase chain reaction process using primers non-selective for thebeta cell specific demethylation pattern, to produce at least twodifferent amplified mixed DNA products having corresponding ratios tothe treated DNA; selectively hybridizing, in a quantitative manner, aplurality of different DNA oligonucleotide probes during the real-timePCR, each respective probe comprising a fluorophore and a quencher,having selectivity for distinguishing between amplified DNA productsfrom the bisulfite-treated DNA having the beta cell specificdemethylation pattern and amplified bisulfite-treated DNA products nothaving beta cell specific demethylation pattern; measuring opticalproperties of the plurality of different DNA oligonucleotide probesduring the real-time PCR, dependent on a change in interaction betweenthe fluorophore and the quencher due to the selectively hybridizing; anddetermining, based on quantitative measurements of the measuredproperties of the plurality of different DNA oligonucleotide probesselectively hybridized to the amplified DNA products from thebisulfite-treated DNA, a ratio of the DNA from cells having the betacell specific demethylation pattern to DNA from cells not having thebeta cell specific demethylation pattern.
 9. The method according toclaim 8, wherein the sample is derived from at least one of bloodplasma, saliva, spinal fluid, lymph fluid, synovial fluid, and tears.10. The method according to claim 9, wherein the polymerase chainreaction is conducted using: a forward primer: SEQ ID NO: 001GTGCGGTTTATATTTGGTGGAAGTT; and a reverse primer: SEQ ID NO: 002ACAACAATAAACAATTAACTCACCCTACAA; and

wherein the plurality of different DNA oligonucleotide probes comprises:a) at least one probe for detection of methylated insulin DNA ofnon-beta cell origin selected from the group consisting of: SEQ ID NO: 3ACCTCCCGACGAATCT; and SEQ ID NO: 4 TACCTCTCGTCGAATCT; and

b) at least one probe for detection of demethylated insulin DNA of betacell origin selected from the group consisting of: SEQ ID NO: 005ACCTCCCAACAAATCT; and SEQ ID NO: 006 TACCTCCCATCAAATCT.


11. The method according to claim 8, wherein the plurality of differentDNA oligonucleotide probes comprises: a) at least one probe fordetection of methylated insulin DNA of non-pancreatic beta cell originselected from the group consisting of: SEQ ID NO: 003 ACCTCCCGACGAATCT;and SEQ ID NO: 004 TACCTCTCGTCGAATCT; and

b) at least one probe for detection of demethylated insulin DNA ofpancreatic beta cell origin selected from the group consisting of:SEQ ID NO: 005 ACCTCCCAACAAATCT; and SEQ ID NO: 006 TACCTCCCATCAAATCT;

and provides a linear relationship between the log ratio of insulin geneDNA of pancreatic beta cell and insulin gene DNA of non-pancreatic betacell origin with an r²=0.98, for DNA samples having a demethylationindex for insulin gene DNA between 100:1 and 1:100; and the plurality ofdifferent DNA oligonucleotide probes provides a detection of DNA fromcells having the cell-type-specific demethylation pattern frompancreatic beta cells subject to 180 fold higher dilution than fromliver or kidney cells.